Spatial/temporal regulation of all-trans-retinoic acid (RA) concentrations, and therefore of RA biosynthesis is crucial during the lifespan of mammals, from development throughout adulthood. The long-term goals of this project are to characterize RA biosynthesis at the biochemical, molecular and endocrine levels. In the last project period, we developed evidence that the physiological substrates of RA biosynthesis and metabolism were the retinoids bound to their specific binding proteins and that retinoids traversed through metabolic pathways via a series of protein-protein interactions between these binding proteins and retinoid-specific enzymes. During this project period, we will test the hypothesis that a major physiological pathway of RA synthesis consists of retinal synthesis from CRBP-retinol (cellular retinol binding protein, type 1) by an NADP-dependent microsomal retinol dehydrogenase, followed by production of RA from CRBP-retinal by a cytosolic NAD-dependent retinal dehydrogenase. The specific aims are: 1) raise antibodies against the cytosolic retinal dehydrogenase and isolate a full-length cDNA; 2) raise antibodies against the microsomal retinol dehydrogenase and isolate a full-length cDNA; 3) physically and kinetically characterize each dehydrogenase and the interactions between each and CRBP; 4) determine immunocytochemically whether CRBP and both dehydrogenases are expressed in the same cells in adult rat tissues, or at the same times and loci during rat development; 5) determine mechanism(s) of regulation of the dehydrogenases by vitamin A status (ie. transcriptional, translational or post-translational) and whether aging affects RA biosynthesis. In addition to testing the hypothesis, the work planned will provide insight into the regulation of RA synthesis, generate fundamental knowledge about developmental and aging affects on RA synthesis, and produce reagents (antibodies, cDNA) for studying endocrine effects on RA synthesis. These reagents will also be useful for identifying diseases that may be caused or exacerbated by altered RA biosynthesis.